The transformation of normal cells to cancer cells is associated with genotypic and/or phenotypic alterations of both the transformed cells and the microenvironment of the growing tumour (Kerbel R S, 1995). Principally, some of these alterations could be recognised by the immune system as tumour specific antigens (TSAs), and thus form the basis for tumour specific immunotherapy. Although mutation events, underlying tumour priming and tumourigenesis, per se may lead to the expression of TSAs, secondary changes like dysregulated expression and posttranslational modifications of normal antigens may include the majority of tumour associated antigens useful as targets for immunotherapy.
Molecules associated with the tumour phenotype can be identified using modern technologies of genomics and proteomics that directly involve the targets themselves for identification of molecular modifications and altered expression levels (Williams K L, 1999). More indirectly, tumour associated antigens (TAAS) characterising the tumour phenotype have also been identified using hybridoma derived monoclonal antibodies (Kohler G. Milstein C, 1976).
The phage display technology has become an established alternative to the hybridoma technology and for some applications even the method of choice for the generation of monoclonal antibodies by an antigen driven selection principle rather than by pure screening (Hoogenboom H R et al, 1998). The technology is based upon bacteriophage particles that on their surface display the particular antibody fragment encoded in their genome allowing the selection of the phage and its encoding DNA as a genetic package. Provided that primers complementary to antibody variable heavy and light chain genes are available, the antibody genes to be inserted in phage vectors can be amplified by polymerase chain reaction (PCR) from immune or non-immune animals of any species, and large phage antibody libraries constructed.
The selection of phage libraries on cells, tissue sections and other biological materials have generated monoclonal antibodies or peptides binding to components within the complex antigen materials used (Hoogenboom H R et al 1998, Tordsson J et al 1997). The outcome from straight positive selections using complex antigens represents an assortment of specificities from the total tissue reacting repertoire, biased towards highly expressed and immunodominant antigens and to high affinity interactions. To rather identify differentially expressed antigens, representative of the specific phenotype, subtractive approaches have to be employed.